Description

WO 2005/040377 PCT/EP2004/011974 HIGH EFFICIENCY GENE TRANSFER AND EXPRESSION IN MAMMALIAN CELLS BY A MULTIPLE TRANSFECTION PROCEDURE OF MAR SEQUENCES FIELD OF THE INVENTION 5 The present invention relates to purified and isolated DNA sequences having protein production increasing activity and more specifically to the use of matrix attachment regions (MARs) for increasing protein production activity in a eukaryotic cell. Also disclosed is a method for the identification of said active regions, in particular MAR 10 nucleotide sequences, and the use of these characterized active MAR sequences in a new multiple transfection method. BACKGROUND OF THE INVENTION 15 Nowadays, the model of loop domain organization of eukaryotic chromosomes is well accepted (Boulikas T, "Nature of DNA sequences at the attachment regions of genes to the nuclear matrix", J. Cell Biochem., 52:14-22, 1993). According to this model chromatin is organized in loops that span 50-100 kb attached to the nuclear matrix, a proteinaceous network made up of RNPs and other nonhistone proteins (Bode J, 20 Stengert-lber M, Kay V, Schalke T and Dietz-Pfeilstetter A, Crit. Rev. Euk. Gene Exp., 6:115-138, 1996). The DNA regions attached to the nuclear matrix are termed SAR or MAR for respectively scaffold (during metaphase) or matrix (interphase) attachment regions 25 (Hart C and Laemmli U (1998), "Facilitation of chromatin dynamics by SARs" Curr Opin Genet Dev 8, 519-525.) As such, these regions may define boundaries of independent chromatin domains, such that only the encompassing cis-regulatory elements control the expression of the 30 genes within the domain. However, their ability to fully shield a chromosomal locus from nearby chromatin elements, and thus confer position-independent gene expression, has not been seen in stably transfected cells (Poljak L, Seum C, Mattioni T and Laemmli U. (1994) "SARs 35 stimulate but do not confer position independent gene expression", Nucleic Acids Res 22, 4386-4394). On the other hand, MAR (or S/MAR) sequences have been shown to interact with enhancers to increase local chromatin accessibility (Jenuwein T, Forrester W, Fernandez-Herrero L, Laible G, Dull M, and Grosschedl R. (1997) "Extension of chromatin accessibility by nuclear matrix attachment regions" Nature 385, 269-272). 40 Specifically, MAR elements can enhance expression of heterologous genes in cell culture lines (Kalos M and Fournier R (1995) "Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chrornatin domain" Mol Cell Biol 15,198-207), transgenic mice (Castilla J, Pintado B, Sola, I, Sanchez-Morgado J, and Enjuanes L (1998) "Engineering passive immunity in 45 transgenic mice secreting virus-neutralizing antibodies in milk" Nat Biotechnol 16, 349 354) and plants (Allen G, Hall GJ, Michalowski S, Newman W, Spiker S, Weissinger A, and Thompson W (1996), "High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco" Plant Cell 8, 899-913). The utility of MAR sequences for developing improved vectors for gene therapy is also recognized 50 (Agarwal M, Austin T, Morel F, Chen J, Bohnlein E, and Plavec 1 (1998), "Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T I CONFIRMATION COPY WO 2005/040377 PCT/EP2004/011974 cells" J Virol 72, 3720-3728). Recently, it has been shown thatchromatin-structure modifying sequences including MARs, as exemplified by the chicken lysozyme 5' MAR is able to significantly enhance 5 reporter expression in pools of stable Chinese Hamster Ovary (CHO) cells (Zahn-Zabal M, et al., "Development of stable cell lines for production or regulated expression using matrix attachment regions" J Biotechnol, 2001, 87(1): p. 29-42). This property was used to increase the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits have been observed both for constructs with 10 MARs flanking the transgene expression cassette, as well as when constructs are co transfected with the MAR on a separate plasmid. However, expression levels upon co transfection with MARs were not as high as those observed for a construct in which two MARs delimit the transgene expression unit. A third and preferable process was shown to be the transfection of transgenes with MARs both linked to the transgene and on a 15 separate plasmid (Girod et al., submitted for publication). However, one persisting limitation of this technique is the quantity of DNA that can be transfected per cell. Many multiples transfection protocols have been developed in order to achieve a high transfection efficiency to characterize the function of genes of interest. The protocol applied by Yamamoto et al, 1999 ("High efficiency gene transfer by multiple transfection 20 protocol", Histochem. J. 31(4), 241-243) leads to a transfection efficiency of about 80 % after 5 transfections events, whereas the conventional transfection protocol only achieved a rate of <40%. While this technique may be useful when one wishes to increase the proportion of expressing cells, it does not lead to cells with a higher intrinsic productivity. Therefore, it cannot be used to generate high producer 25 monoclonal cell lines. Hence, the previously described technique has two major drawbacks: i) this technique does not generate a homogenous population of transfected cells, since it cannot favour the integration of further gene copy, nor does it direct the transgenes to favorable chromosomal loci, 30 ii) the use of the same selectable marker in multiple transfection events does not permit the selection of doubly or triply transfected cells. In patent application WO02/074969, the utility of MARs for the development of stable eukaryotic cell lines has also been demonstrated. However, this application does not 35 disclose neither any conserved homology for MAR DNA element nor any technique for predicting the ability for a DNA sequence to be a MAR sequence. In fact no clear-cut MAR consensus sequence has been found (Boulikas T, "Nature of DNA sequences at the attachment regions of genes to the nuclear matrix", J. Cell 40 Biochem., 52:14-22, 1993) but evolutionarily, the structure of these sequences seem to be functionally conserved in eukaryotic genomes, since animal MARs can bind to plant nuclear scaffolds and vice versa (Mielke C, Kohwi Y, Kohwi-Shigematsu T and Bode J, "Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo", Biochemistry, 29:7475-7485, 45 1990). The identification of MARs by biochemical studies is a long and unpredictable process; various results can be obtained depending on the assay (Razin SV, "Functional architecture of chromosomal DNA domains", Crit Rev Eukaryot Gene Expr., 6:247-269, 50 1996). Considering the huge number of expected MARs in a eukaryotic genome and the amount of sequences issued from genome projects, a tool able to filter potential MARS in order to perform targeted experiments would be greatly useful. 2 WO 2005/040377 PCT/EP2004/011974 Currently two different predictive tools for MARs are available via the Internet. The fist one, MAR-Finder (http://futuresoft.org/MarFinder; Singh GB, Kramer JA and Krawetz SA, "Mathematical model to predict regions of chromatin attachment to the nuclear matrix", Nucleic Acid Research, 25:1419-1425, 1997) is based on set of 5 patterns identified within several MARs and a statistical analysis of the co-occurrence of these patterns. MAR-Finder predictions are dependent of the sequence context, meaning that predicted MARs depend on the context of the submitted sequence. The other predictive software, SMARTest (http://www. genomatix.de; Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I and Werner T, "In silico prediction of 10 scaffold/matrix attachment regions in large genomic sequences", Genome Research, 12:349-354, 2001), use weight-matrices derived from experimentally identified MARs. SMARTest is said to be suitable to perform large-scale analyses. But actually aside its relative poor specificity, the amount of hypothetical MARs rapidly gets huge when doing large scale analyses with it, and in having no way to increase its specificity to restrain 15 the number of hypothetical MARs, SMARTest becomes almost useless to screen for potent MARs form large DNA sequences. Some other softwares, not available via the Internet, also exists; they are based as well on the frequency of MAR motifs (MRS criterion;Van Drunen CM et al., "A bipartite sequence element associated with matrix/scaffold attachment regions", Nucleic Acids 20 Res, 27:2924-2930, 1999), (ChrClass; Glazko GV et al., "Comparative study and prediction of DNA fragments associated with various elements of the nuclear matrix", Biochim. Biophys. Acta, 1517:351-356, 2001) or based on the identification of sites of stress-induced DNA duplex (SIDD; Benham C and al., "Stress-induced duplex DNA destabilization in scaffold/matrix attachment regions", J. Mol. Biol., 274:181-196, 1997). 25 However, their suitability to analyze complete genome sequences remains unknown, and whether these tools may allow the identification of protein production-increasing sequences has not been reported. Furthermore, due to the relatively poor specificity of these softwares (Frisch M, Frech K, 30 Klingenhoff A, Cartharius K, Liebich I and Werner T, "In silico prediction of scaffold/matrix attachment regions in large genomic sequences", Genome Research, 12:349-354, 2001), the amount of hypothetical MARs identified in genomes rapidly gets unmanageable when doing large scale analyses, especially if most of these have no or poor activity in practice. Thus, having no way to increase prediction specificity to 35 restrain the number of hypothetical MARs, many of the available programs become almost useless to identify potent genetic elements in view of efficiently increasing recombinant protein production. Since all the above available predictive methods have some drawbacks that prevent 40 large-scale analyses of genomes to identify reliably novel and potent MARs, the object of this invention is to 1) understand the functional features of MARs that allow improved recombinant protein expression; 2) get a new Bioinformatic tool compiling MAR structural features as a prediction of function, in order to 3) perform large scale analyses of genomes to identify novel and more potent MARs, and, finally 4) to 45 demonstrate improved efficiency to increase the production of recombinant proteins from eukaryotic cells or organisms when using the newly identified MAR sequences. SUMMARY OF THE INVENTION 50 This object has been achieved by providing an improved and reliable method for the identification of DNA sequences having protein production increasing activity, in 3 WO 2005/040377 PCT/EP2004/011974 particular MAR nucleotide sequences, and the use of these characterized active MAR sequences in a new multiple transfection method to increase the production of recombinant proteins in eukaryotic cells. 5 BRIEF DESCRIPTION OF THE FIGURES Fig. I shows the distribution plots of MARs and non-MARs sequences. Histograms are density plots (relative frequency divided by the bin width) relative to the score of the observed parameter. The density histogram for human MARs in the SMARt DB 10 database is shown in black, while the density histogram for the human chromosome 22 are in grey. Fig. 2 shows Scatterplots of the four different criteria used by SMAR Scan@ and the AT-content with human MARs from SMARt DB. 15 Fig. 3 shows the distribution plots of MAR sequences by organism. MAR sequences from SMARt DB of other organisms were retrieved and analyzed. The MAR sequences density distributions for the mouse, the chicken, the sorghum bicolor and the human are plotted jointly. 20 Fig. 4 shows SMAR Scan@ predictions on human chromosome 22 and on shuffled chromosome 22. Top plot : Average number of hits obtained by SMAR Scan@ with five: rubbled, scrambled, shuffled within nonoverlapping windows of 10 bp, order 1 Markov chains model and with the native chromosome 22. Bottom plot: Average number of 25 MARs predicted by SMAR Scan@ in five: rubbled, scrambled, shuffled within non overlapping windows of 10 bp, order 1 Markov chains model and with the native chromosome 22. Fig. 5 shows the dissection of the ability of the chicken lysozyme gene 5'-MAR to 30 stimulate transgene expression in CHO-DG44 cells. Fragments B, K and F show the highest ability to stimulate transgene expression. The indicated relative strength of the elements was based on the number of high-expressor cells. Fig. 6 shows the effect of serial-deletions of the 5'-end (upper part) and the 3'-end 35 (lower part) of the 5'-MAR on the loss of ability to stimulate transgene expression. The transition from increased to decreased activity coincide with B-, K- and F-fragments. Fig. 7 shows that portions of the F fragment significantly stimulate transgene expression. The F fragment regions indicated by the light grey arrow were multimerized, 40 inserted in pGEGFP Control and transfected in CHO cells. The element that displays the highest activity is located in the central part of the element and corresponds to fragment Fill (black bar labelled minimal MAR). In addition, an enhancer activity is located in the 3'-flanking part of the Fill fragment (dark grey bar labelled MAR enhancer). 45 Fig. 8 shows a map of locations for various DNA sequence motifs within the cLysMAR. Fig. 8 (B) represents a Map of locations for various DNA sequence motifs within the cLysMAR. Vertical lines represent the position of the computer-predicted sites or sequence motifs along the 3034 base pairs of the cLysMAR and its active regions, as 50 presented in Fig. 5. The putative transcription factor sites, (MEF2 05, Oct-1, USF-02, GATA, NFAT) for activators and (CDP, SATB1, CTCF, ARBP/MeCP2) for repressors of transcription, were identified using Matinspector (Genomatix), and CpG islands were identifed with CPGPLOT. Motifs previously associated with MAR elements are labelled 4 WO 2005/040377 PCT/EP2004/011974 in black and include CpG dinucleotides and CpG islands, unwinding motifs (AATATATT and AATATT), poly As and Ts, poly Gs and Cs, Drosophila topoisomerase II binding sites (GTNWAYATTNATTNATNNR) which had identity to the 6 bp core and High mobility group I (HMG-l/Y) protein binding sites. Other structural motifs include 5 nucleosome-binding and nucleosome disfavouring sites and a motif thought to relieve the superhelical strand of DNA. Fig. 8(A) represents the comparison of the ability of portions of the cLysMAR to activate transcription with MAR prediction score profiles with MarFinder. The top diagram shows the MAR fragment activity as in Fig. 5, while the middle and bottom curves show MARFinder-predicted potential for MAR activity and 10 for bent DNA structures respectively. Fig. 9 shows the correlation of DNA physico-chemical properties with MAR activity. Fig. 9(A), represents the DNA melting temperature, double helix bending, major groove depth and minor groove width profiles of the 5'-MAR and were determined using the 15 algorithms of Levitsky et al (Levitsky VG, Ponomarenko MP, Ponomarenko JV, Frolov AS, Kolchanov NA "Nucleosomal DNA property database", Bioinformatics, 15; 582592, 1999). The most active B, K and F fragments depicted at the top are as shown as in Figure 1. Fig. 9(B), represents the enlargement of the data presented in panel A to display the F fragment map aligned with the tracings corresponding to the melting 20 temperature (top curve) and DNA bending (bottom curve). The position of the most active FIB fragment and protein binding site for specific transcription factors are as indicated. Fig. 10 shows the distribution of putative transcription factor binding sites within the 5' 25 cLysMAR. Large arrows indicate the position of the CUE elements as identified with SMAR Scan@. Fig. 11 shows the scheme of assembly of various portions of the MAR. The indicated portions of the cLysMAR were amplified by PCR, introducing Bglll-BamHI linker 30 elements at each extremity, and assembled to generate the depicted composite elements. For instance, the top construct consists of the assembly of all CUE and flanking sequences at their original location except that BgIl-BamHll linker sequences separate each element. 35 Fig. 12 represents the plasmid maps. Fig. 13 shows the effect of re-transfecting primary transfectants on GFP expression. Cells (CHO-DG44) were co-transfected with pSV40EGFP (left tube) or pMAR SV40EGFP (central tube) and pSVneo as resistance plasmid. Cells transfected with 40 pMAR-SV40EGFP were re-transfected 24 hours later with the same plasmid and a different selection plasmid, pSVpuro (right tube). After two weeks selection, the phenotype of the stably transfected cell population was analysed by FACS. Fig. 14 shows the effect of multiple load of MAR-containing plasmid. The pMAR 45 SV40EGFP/ pMAR-SV40EGFP secondary transfectants were used in a third cycle of transfection at the end of the selection process. The tertiary transfection was accomplished with pMAR or pMAR-SV40EGFP to give tertiary transfectants. After 24 hours, cells were transfected again with either plasmid, resulting in the quaternary transfectants (see Table 4). 50 Fig. 15 shows comparative performance of SMAR prediction algorithms exemplified by region WP18A10A7. (A) SMAR Scan@ analysis was performed with default settings. (B) SIDD analysis (top curve and left-hand side scale), and the attachment of several 5 WO 2005/040377 PCT/EP2004/011974 DNA fragments to the nuclear matrix in vitro (bar-graph, right-hand side scale) was taken from Goetze et al ( Goetze S, Gluch A, Benham C, Bode J, "Computational and in vitro analysis of destabilized DNA regions in the interferon gene cluster: potential of predicting functional gene domains." Biochemistry, 42:154-166, 2003). 5 Fig. 16 represents the results of a a gene therapy-like protocol using MARs. The group of mice injected by MAR-network, induced from the beginning of the experiment, display a better induction of the hematocrit in comparison of mice injected by original network without MAR. After 2 months, hematocrits in "MAR-containing 10 group" is still at values higher (65%) than normal hematocrit levels (45-55%). Fig. 17 represents the scatterplot for the 1757 S/MAR sequences of the AT (top) and TA (bottom) dinucleotide percentages versus the predicted DNA bending as computed by SMAR Scan@. 15 Fig. 18 represents the dinucleotide percentage distribution plots over the 1757 non S/MARs sequences. 20 Fig.19 shows the effect of various S/MAR elements on the production of recombinant green fluorescent protein (GFP). Populations of CHO cells transfected with a GFP expression vector containing or a MAR element, as indicated, were analyzed by a fluorescence-activated cell sorter (FACS®), and typical profiles are shown. The profiles display the cell number counts as a function of the GFP fluorescence levels. 25 Fig. 20 depicts the effect of the induction of hematocrit in mice injected by MAR network. 30 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a purified and isolated DNA sequence having protein production increasing activity characterized in that said DNA sequence comprises at least one bent DNA element, and at least one binding site for a DNA binding protein. 35 Certain sequences of DNA are known to form a relatively "static curve", where the DNA follows a particular 3-dimensional path. Thus, instead of just being in the normal B DNA conformation ("straight"), the piece of DNA can form a flat, planar curve also defined as bent DNA (Marini, et al., 1982 "Bent helical structure in kinetoplast DNA", 40 Proc. Nati. Acad. Sci. USA, 79: 7664-7664). Surprisingly, Applicants have shown that the bent DNA element of a purified and isolated DNA sequence having protein production increasing activity of the present invention usually contains at least 10% of dinucleotide TA, and/or at least 12% of 45 dinucleotide AT on a stretch of 100 contiguous base pairs. Preferably, the bent DNA element contains at least 33% of dinucleotide TA, and/or at least 33% of dinucleotide AT on a stretch of 100 contiguous base pairs. These data have been obtained by the method described further. 50 According to the present invention, the purified and isolated DNA sequence usually comprises a MAR nucleotide sequence selected from the group comprising the sequences SEQ ID Nos 1 to 27 or a cLysMAR element or a fragment thereof. Preferably, the purified and isolated DNA sequence is a MAR nucleotide sequence 6 selected from the group comprising the sequences SEQ ID Nos1 to 27, more preferably the sequences SEQ ID Nos 24 to 27. There is therefore provided a purified and isolated DNA sequence having protein production increasing activity comprising: 5 a) at least one bent DNA element, and b) at least one binding site for a DNA binding protein, wherein said DNA sequence is a MAR nucleotide with a sequence selected from the following: 0 (i) any one of SEQ ID NOs: 24-27, (ii) a sequence complementary to any one of SEQ ID NOs: 24-27, (iii) a part of any one of SEQ ID NOs: 24-27 sharing at least 70% nucleotides in length, (iv) a molecular chimera of any one of SEQ ID NOs: 24-27, 5 (v) a combination of any of (i) to (iv), or (vi) variants of any of (i) to (v). Encompassed by the present invention are as well complementary sequences of the above-mentioned sequences SEQ ID Nos 1 to 27 and the cLysMAR element or fragment, which can be produced by using PCR or other means. 20 An "element" is a conserved nucleotide sequences that bears common functional properties (i.e. binding sites for transcription factors) or structural (i.e. bent DNA sequence) features. A part of sequences SEQ ID Nos 1 to 27 and the cLysMAR element or fragment refers to sequences sharing at least 70% nucleotides in length with the respective 25 sequence of the SEQ ID Nos 1 to 27. These sequences can be used as long as they exhibit the same properties as the native sequence from which they derive. Preferably these sequences share more than 80%, in particular more than 90% nucleotides in length with the respective sequence of the SEQ ID Nos 1 to 27. The present invention also includes variants of the aforementioned sequences SEQ 7/1 ID Nos 1 to 27 and the cLysMAR element or fragment, that is nucleotide sequences that vary from the reference sequence by conservative nucleotide substitutions, whereby one or more nucleotides are substituted by another with the same characteristics. s The sequences SEQ ID Nos 1 to 23 have been identified by scanning human chromosome 1 and 2 using SMAR Scan®, showing that the identification of novel MAR sequenced is feasible using the tools reported thereafter whereas SEQ ID No 24 to 27 have been identified by scanning the complete human genome using the combined SMAR Scan® method. o In a first step, the complete chromosome 1 and 2 were screened to identify bent DNA element as region corresponding to the highest bent, major groove depth, minor groove width and lowest melting temperature as shown in figure 3. In a second step, this collection of sequence was scanned for binding sites or regulatory proteins such as SATB1, GATA, etc. as shown in the figure 8B) yielding sequences 5 SEQ ID 1-23. Furthermore, sequences 21-23 were further shown to be located next to known gene from the Human Genome Data Base. With regard to SEQ ID No 24 to 27 these sequences have been yielded by scanning the human genome according to the combined method and were selected as examples among 1757 MAR elements so detected. o Molecular chimera of MAR sequences are also considered in the present invention. By molecular chimera is intended a nucleotide sequence that may include a functional portion of a MAR element and that will be obtained by molecular biology methods known by those skilled in the art. Particular combinations of MAR elements or fragments or sub-portions thereof are 25 also considered in the present invention. These fragments can be prepared by a variety of methods known in the art. These methods include, but are not limited to, digestion with restriction enzymes and recovery of the fragments, chemical synthesis or polymerase chain reactions (PCR). Therefore, particular combinations of elements or fragments of the SEQ ID 7/2 WO 2005/040377 PCT/EP2004/011974 Nos 1 to 27 and cLysMAR elements or fragments are also envisioned in the present invention, depending on the functional results to be obtained. Elements of the cLysMAR are e.g. the B, K and F regions as described in WO 02/074969, the disclosure of which is hereby incorporated herein by reference, in its entirety. The preferred elements of the 5 cLysMAR used in the present invention are the B, K and F regions. Only one element might be used or multiple copies of the same or distinct elements (multimerized elements) might be used (see Fig. 8 A)). By fragment is intended a portion of the respective nucleotide sequence. Fragments of 10 a MAR nucleotide sequence may retain biological activity and hence bind to purified nuclear matrices and/or alter the expression patterns of coding sequences operably linked to a promoter. Fragments of a MAR nucleotide sequence may range from at least about 100 to 1000 bp, preferably from about 200 to 700 bp, more preferably from about 300 to 500 bp nucleotides. Also envisioned are any combinations of fragments, 15 which have the same number of nucleotides present in a synthetic MAR sequence consisting of natural MAR element and/or fragments. The fragments are preferably assembled by linker sequences. Preferred linkers are Bglll-BamHl linker. "Protein production increasing activity" refers to an activity of the purified and isolated 20 DNA sequence defined as follows: after having been introduced under suitable conditions into a eukaryotic host cell, the sequence is capable of increasing protein production levels in cell culture as compared to a culture of cell transfected without said DNA sequence. Usually the increase is 1.5 to 10 fold, preferably 4 to 10 fold. This corresponds to a production rate or a specific cellular productivity of at least 10 pg per 25 cell per day (see Example 11 and Fig.13). As used herein, the following definitions are supplied in order to facilitate the understanding of this invention. 30 "Chromatin" is the protein and nucleic acid material constituting the chromosomes of a eukaryotic cell, and refers to DNA, RNA and associated proteins. A "chromatin element" means a nucleic acid sequence on a chromosome having the property to modify the chromatine structure when integrated into that chromosome. 35 "Cis" refers to the placement of two or more elements (such as chromatin elements) on the same nucleic acid molecule (such as the same vector, plasmid or chromosome). "Trans" refers to the placement of two or more elements (such as chromatin elements) 40 on two or more different nucleic acid molecules (such as on two vectors or two chromosomes). Chromatin modifying elements that are potentially capable of overcoming position effects, and hence are of interest for the development of stable cell lines, include 45 boundary elements (BEs), matrix attachment regions (MARs), locus control regions (LCRs), and universal chromatin opening elements (UCOEs). Boundary elements ("BEs"), or insulator elements, define boundaries in chromatin in many cases (Bell A and Felsenfeld G. 1999; "Stopped at the border: boundaries and 50 insulators, Curr Opin Genet Dev 9, 191-198) and may play a role in defining a transcriptional domain in vivo. BEs lack intrinsic promoter/enhancer activity, but rather are thought to protect genes from the transcriptional influence of regulatory elements in the surrounding chromatin. The enhancer-block assay is commonly used to identify 8 WO 2005/040377 PCT/EP2004/011974 insulator elements. In this assay, the chromatin element is placed between an enhancer and a promoter, and enhancer-activated transcription is measured. Boundary elements have been shown to be able to protect stably transfected reporter genes against position effects in Drosophila, yeast and in mammalian cells. They have also been 5 shown to increase the proportion of transgenic mice with inducible transgene expression. Locus control regions ("LCRs") are cis-regulatory elements required for the initial chromatin activation of a locus and subsequent gene transcription in their native 10 locations (Grosveld, F. 1999, "Activation by locus control regions?" Curr Opin Genet Dev 9, 152-157). The activating function of LCRs also allows the expression of a coupled transgene in the appropriate tissue in transgenic mice, irrespective of the site of integration in the host genome. While LCRs generally confer tissue-specific levels of expression on linked genes, efficient expression in nearly all tissues in transgenic mice 15 has been reported for a truncated human T-cell receptor LCR and a rat LAP LCR. The most extensively characterized LCR is that of the globin locus. Its use in vectors for the gene therapy of sickle cell disease and (3-thalassemias is currently being evaluated. "MARs", according to a well-accepted model, may mediate the anchorage of specific 20 DNA sequence to the nuclear matrix, generating chromatin loop domains that extend outwards from the heterochromatin cores. While MARs do not contain any obvious consensus or recognizable sequence, their most consistent feature appears to be an overall high A/T content, and C bases predominating on one strand (Bode J, Schlake T, RiosRamirez M, Mielke C, Stengart M, Kay V and KlehrWirth D, "Scaffold/matrix 25 attached regions: structural propreties creating transcriptionally active loci",Structural and Functional Organization of the Nuclear Matrix: International Review of Citology, 162A:389453, 1995). These regions have a propensity to form bent secondary structures that may be prone to strand separation. They are often referred to as base unpairing regions (BURs), and they contain a core-unwinding element (CUE) that might 30 represent the nucleation point of strand separation (Benham C and al., Stress induced duplex DNA destabilization in scaffold/matrix attachment regions, J. Mol. Biol., 274:181 196, 1997). Several simple AT-rich sequence motifs have often been found within MAR sequences, but for the most part, their functional importance and potential mode of action remain unclear. These include the A-box (AATAAAYAAA), the T-box 35 (TTWTWTTWTT), DNA unwinding motifs (AATATATT, AATATT), SATB1 binding sites (H-box, A/T/C25) and consensus Topoisomerase Il sites for vertebrates (RNYNNCNNGYNGKTNYNY) or Drosophila (GTNWAYATTNATNNR). Ubiquitous chromatin opening elements ("UCOEs", also known as "ubiquitously-acting 40 chromatin opening elements") have been reported in WO 00/05393. An "enhancer" is a nucleotide sequence that acts to potentiate the transcription of genes independent of the identity of the gene, the position of the sequence in relation to the gene, or the orientation of the sequence. The vectors of the present invention 45 optionally include enhancers. A "gene" is a deoxyribonucleotide (DNA) sequence coding for a given mature protein. As used herein, the term "gene" shall not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or 50 enhancers. A "product gene" is a gene that encodes a protein product having desirable characteristics such as diagnostic or therapeutic utility. A product gene includes, e. g., 9 WO 2005/040377 PCT/EP2004/011974 structural genes and regulatory genes. A "structural gene" refers to a gene that encodes a structural protein. Examples of structural genes include but are not limited to, cytoskeletal proteins, extracellular matrix 5 proteins, enzymes, nuclear pore proteins and nuclear scaffold proteins, ion channels and transporters, contractile proteins, and chaperones. Preferred structural genes encode for antibodies or antibody fragments. A "regulatory gene" refers to a gene that encodes a regulatory protein. Examples of 10 regulatory proteins include, but are not limited to, transcription factors, hormones, growth factors, cytokines, signal transduction molecules, oncogenes, proto-oncogenes, transmembrane receptors, and protein kinases. "Orientation" refers to the order of nucleotides in a given DNA sequence. For example, 15 an inverted orientation of a DNA sequence is one in which the 5' to 3' order of the sequence in relation to another sequence is reversed when compared to a point of reference in the DNA from which the sequence was obtained. Such reference points can include the direction of transcription of other specified DNA sequences in the source DNA and/or the origin of replication of replicable vectors containing the 20 sequence. "Eukaryotic cell" refers to any mammalian or non-mammalian cell from a eukaryotic organism. By way of non-limiting example, any eukaryotic cell that is capable of being maintained under cell culture conditions and subsequently transfected would be 25 included in this invention. Especially preferable cell types include, e. g., stem cells, embryonic stem cells, Chinese hamster ovary cells (CHO), COS, BHK21, NIH3T3, HeLa, C2C12, cancer cells, and primary differentiated or undifferentiated cells. Other suitable host cells are known to those skilled in the art. 30 The terms "host cell" and "recombinant host cell" are used interchangeably herein to indicate a eukaryotic cell into which one or more vectors of the invention have been introduced. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or 35 environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The terms "introducing a purified DNA into a eukaryotic host cell" or "transfection" denote any process wherein an extracellular DNA, with or without accompanying 40 material, enters a host cell. The term "cell transfected" or "transfected cell" means the cell into which the extracellular DNA has been introduced and thus harbours the extracellular DNA. The DNA might be introduced into the cell so that the nucleic acid is replicable either as a chromosomal integrant or as an extra chromosomal element. 45 "Promoter" as used herein refers to a nucleic acid sequence that regulates expression of a gene. "Co-transfection" means the process of transfecting a eukaryotic cell with more than one exogenous gene, or vector, or plasmid, foreign to the cell, one of which may confer 50 a selectable phenotype on the cell. 10 WO 2005/040377 PCT/EP2004/011974 The purified and isolated DNA sequence having protein production increasing activity also comprises, besides one or more bent DNA element, at least one binding site for a DNA binding protein. 5 Usually the DNA binding protein is a transcription factor. Examples of transcription factors are the group comprising the polyQpolyP domain proteins. Another example of a transcription factor is a transcription factor selected from the group comprising SATB1, NMP4, MEF2, S8, DLX1, FREAC7, BRN2, GATA 1/3, TATA, Bright, MSX, AP1, C/EBP, CREBP1, FOX, Freac7, HFH1, HNF3alpha, Nkx25, 10 POU3F2, Pit1, TTFI, XFDI, AR, C/EBPgamma, Cdc5, FOXD3, HFH3, HNF3 beta, MRF2, Oct1, POU6FI, SRF, V$MTATA_B, XFD2, Bach2, CDP CR3, Cdx2, FOXJ2, HFL, HP1, Myc, PBX, Pax3, TEF, VBP, XFD3, Brn2, COMPI, Evil, FOXP3, GATA4, HFNI, Lhx3, NKX3A, POUIF1, Pax6, TFIIA or a combination of two or more of these transcription factors are preferred. Most preferred are SATB1, NMP4, MEF2 and 15 polyQpolyP domain proteins. SATB1, NMP4 and MEF2, for example, are known to regulate the development and/or tissue-specific gene expression in mammals. These transcription factors have the capacity to alter DNA geometry, and reciprocally, binding to DNA as an allosteric ligand 20 modifies their structure. Recently, SATB1 was found to form a cage-like structure circumscribing heterochromatin (Cai S, Han HJ , and Kohwi-Shigematsu T, "Tissue specific nuclear architecture and gene expression regulated by SATB1" Nat Genet, 2003. 34(1): p. 42-51). 25 Yet another object of the present invention is to provide a purified and isolated cLysMAR element and/or fragment, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. 30 More preferably, the cLysMAR element and/or fragment are consisting of at least one nucleotide sequence selected from the B, K and F regions. A further object of the present invention is to provide a synthetic MAR sequence comprising natural MAR element and/or fragments assembled between linker 35 sequences. Preferably, the synthetic MAR sequence comprises a cLysMAR element and/or fragment a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. 40 Also preferably, linker sequences are Bglll-BamHl linker. An other aspect of the invention is to provide a method for identifying a MAR sequence using a Bioinformatic tool comprising the computing of values of one or more DNA sequence features corresponding to DNA bending, major groove depth and minor 45 groove width potentials and melting temperature. Preferably, the identification of one or more DNA sequence features further comprises a further DNA sequence feature corresponding to binding sites for DNA binding proteins, which is also computed with this method. 50 Preferably, profiles or weight-matrices of said bioinformatic tool are based on dinucleotide recognition. 11 WO 2005/040377 PCT/EP2004/011974 The bioinformatic tool used for the present method is preferably, SMAR Scan@, which contains algorithms developed by Gene Express (http://srs6.bionet.nsc.ru/srs6bin/cgi bin/wqetz?-e+[FEATURES-SitelD:'nR']) and based on Levitsky et al., 1999. These algorithms recognise profiles, based on dinucleotides weight-matrices, to compute the 5 theoretical values for conformational and physicochemical properties of DNA. Preferably, SMAR Scan@ uses the four theoretical criteria also designated as DNA sequence features corresponding to DNA bending, major groove depth and minor groove width potentials, melting temperature in all possible combination, using scanning 10 windows of variable size (see Fig. 3). For each function used, a cut-off value has to be set. The program returns a hit every time the computed score of a given region is above the set cut-off value for all of the chosen criteria. Two data output modes are available to handle the hits, the first (called "profile-like") simply returns all hit positions on the query sequence and their corresponding values for the different criteria chosen. The 15 second mode (called "contiguous hits ") returns only the positions of several contiguous hits and their corresponding sequence. For this mode, the minimum number of contiguous hits is another cut-off value that can be set, again with a tunable window size. This second mode is the default mode of SMAR Scan@. Indeed, from a semantic point of view, a hit is considered as a core-unwinding element (CUE), and a cluster of 20 CUEs accompanied by clusters of binding sites for relevant proteins is considered as a MAR. Thus, SMAR Scan@ considers only several contiguous hits as a potential MAR. To tune the default cut-off values for the four theoretical structural criteria, experimentally validated MARs from SMARt DB (http://transfac.gbf.de/- SMARt DB) 25 were used. All the human MAR sequences from the database were retrieved and analyzed with SMAR Scan@ using the "profile-like" mode with the four criteria and with no set cut-off value. This allowed the setting of each function for every position of the sequences. The distribution for each criterion was then computed according to these data (see Fig. 1 and 3). 30 The default cut-off values of SMAR Scan@ for the bend, the major groove depth and the minor groove width were set at the average of the 75th quantile and the median. For the melting temperature, the default cut-off value should be set at the 75th quantile. The minimum length for the "contiguous-hits" mode should be set to 300 because it is 35 assumed to be the minimum length of a MAR (see Fig. 8 and 9). However, one skilled in the art would be able to determine the cut-off values for the above-mentioned criteria for a given organism with minimal experimentation. 40 Preferably, DNA bending values are comprised between 3 to 5 0 (radial degree). Most preferably they are situated between 3.8 to 4.4 0, corresponding to the smallest peak of Fig. 1. Preferably the major groove depth values are comprised between 8.9 to 9.3 A 45 (Angstr6m) and minor groove width values between 5.2 to 5.8 A. Most preferably the major groove depth values are comprised between 9.0 to 9.2 A and minor groove width values between 5.4 to 5.7 A. Preferably the melting temperature is comprised between 55 to 75 0 C (Celsius degree). 50 Most preferably, the melting temperature is comprised between 55 to 62 0 C. The DNA binding protein of which values can be computed by the method is usually a transcription factor preferably a polyQpolyP domain or a transcription factor selected 12 WO 2005/040377 PCT/EP2004/011974 from the group comprising SATB1, NMP4, MEF2, S8, DLX1, FREAC7, BRN2, GATA 1/3, TATA, Bright, MSX, AP1, C/EBP, CREBP1, FOX, Freac7, HFH1, HNF3alpha, Nkx25, POU3F2, Pit1, TTF1, XFD1, AR, C/EBPgamma, Cdc5, FOXD3, HFH3, HNF3 beta, MRF2, Oct1, POU6F1, SRF, V$MTATA_B, XFD2, Bach2, CDP CR3, Cdx2, 5 FOXJ2, HFL, HP1, Myc, PBX, Pax3, TEF, VBP, XFD3, Brn2, COMP1, Evil, FOXP3, GATA4, HFN1, Lhx3, NKX3A, POUI F1, Pax6, TFIIA or a combination of two or more of these transcription factors. However, one skilled in the art would be able to determine other kinds of transcription 10 factors in order to carry out the method according to the present invention. In case SMAR Scan@ is envisaged to perform, for example, large scale analysis, then, preferably, the above-mentioned method further comprises at least one filter predicting 15 DNA binding sites for DNA transcription factors in order to reduce the computation. The principle of this method combines SMAR Scan@ to compute the structural features as described above and a filter, such as for example, the pfsearch, (from the pftools package as described in Bucher P, Karplus K, Moeri N, and Hofmann K, "A flexible 20 search technique based on generalized profiles", Computers and Chemistry, 20:324, 1996) to predict the binding of some transcription factors. Examples of filters comprise, but are not limited to, pfsearch, MatInspector, RMatch Professional and TRANSFAC Professional 25 This combined method uses the structural features of SMAR Scan@ and the predicted binding of specific transcription factors of the filter that can be applied sequentially in any order to select MARs, therefore, depending on the filter is applied at the beginning or at the end of the method. 30 The first level selects sequences out of the primary input sequence and the second level, consisting in the filter, may be used to restrain among the selected sequences those which satisfy the criteria used by the filter. 35 In this combined method the filter detects clusters of DNA binding sites using profiles or weightmatrices from, for example, Matinspector (Quandt K, Frech K, Karas H, Wingender E, Werner T, "MatInd and MatInspector New fast and versatile tools for detection of consensus matches in nucleotide sequence data", Nucleic Acids Research , 23, 48784884, 1995.). The filter can also detect densities of clusters of DNA binding 40 sites. The combined method is actually a "wrapper" written in PerI for SMAR Scan@ and, in case the pfsearch is used as a filter, from the pftools. The combined method performs a twolevel processing using at each level one of these tools (SMAR Scan@ or filter) as a 45 potential "filter", each filter being optional and possible to be used to compute the predicted features without doing any filtering. If SMAR Scan@ is used in the first level to filter subsequences, it has to be used with the "all the contiguous hits" mode in order to return sequences. If the pfsearch is used 50 in the first level as first filter, it has to be used with only one profile and a distance in nucleotide needs to be provided. This distance is used to group together pfsearch hits that are located at a distance inferior to the distance provided in order to return sequences; The combined method launches pfsearch, parses its output and returns 13 WO 2005/040377 PCT/EP2004/011974 sequences corresponding to pfsearch hits that are grouped together according to the distance provided. Then whatever the tool used in the first level, the length of the sub sequences thus selected can be systematically extended at both ends according to a parameter called "hits extension". 5 The second and optional level can be used to filter out sequences (already filtered sequences or unfiltered input sequences) or to get the results of SMAR Scan@ and/or pfsearch without doing any filtering on these sequences. If the second level of combined method is used to filter, for each criteria considered cutoff values (hit per 10 nucleotide)need to be provided to filter out those sequences (see Fig. 20). Another concern of the present invention is also to provide a method for identifying a MAR sequence comprising at least one filter detecting clusters of DNA binding sites using profiles or weightmatrices. Preferably, this method comprises two levels of filters 15 and in this case, SMAR Scan@ is totally absent from said method. Usually, the two levels consist in pfsearch. Also embraced by the present invention is a purified and isolated MAR DNA sequence identifiable according to the method for identifying a MAR sequence using the 20 described bioinformatic tool, the combined method or the method comprising at least one filter. Analysis by the combined method of the whole human genome yielded a total of 1757 putative MARs representing a total of 1 065 305 base paires. In order to reduce the number of results, a dinucleotide analysis was performed on these 1757 MARs, 25 computing each of the 16 possible dinucleotide percentage for each sequence considering both strands in the 5' to 3' direction. Surprisingly, Applicants have shown that all of the "super" MARs detected with the combined method contain at least 10% of dinucleotide TA on a stretch of 100 30 contiguous base pairs. Preferably, these sequences contain at least 33% of dinucleotide TA on a stretch of 100 contiguous base pairs. Applicants have also shown that these same sequences further contain at least 12% of dinucleotide AT on a stretch of 100 contiguous base pairs. Preferably, they contain at 35 least 33% of dinucleotide AT on a stretch of 100 contiguous base pairs. An other aspect of the invention is to provide a purified and isolated MAR DNA sequence of any of the preceding described MARs, comprising a sequence selected from the sequences SEQ ID Nos 1 to 27, a sequence complementary thereof, a part 40 thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. Preferably, said purified and isolated MAR DNA sequence comprises a sequence selected from the sequences SEQ ID Nos 24 to 27, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera 45 thereof, a combination thereof and variants. These sequences 24 to 27 correspond to those detected by the combined method and show a higher protein production increasing activity over sequences 1 to 23. The present invention also encompasses the use of a purified and isolated DNA 50 sequence comprising a first isolated matrix attachment region (MAR) nucleotide sequence which is a MAR nucleotide sequence selected from the group comprising 14 - a purified and isolated DNA sequence having protein production increasing activity, - a purified and isolated MAR DNA sequence identifiable according to the method for identifying a MAR sequence using the described bioinformatic tool, the combined method or the method comprising at least one filter, - the sequences SEQ ID Nos 1 to 27, - a purified and isolated cLysMAR element and/or fragment, - a synthetic MAR sequence comprising natural MAR element and/or fragments assembled between linker sequences, o a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants or a MAR nucleotide sequence of a cLysMAR element and/or fragment, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants for increasing protein 5 production activity in a eukaryotic host cell. There is further provided the use of a purified and isolated DNA sequence comprising a first isolated matrix attachment region (MAR) nucleotide sequence selected from the group comprising: o (i) a purified and isolated DNA sequence of claims 1 to 6, (ii) the sequence any one of SEQ ID NOs: 24-27, (iii) a synthetic MAR sequence of claims 7 or 8, (iv) a sequence complementary to any one of (i) to (iii), (v) a part of any one of claims (i) to (iii) sharing at least 70% nucleotides in 25 length, (vi) a molecular chimera of any one of claims (i) to (iii), (vii) a combination of any one of claims (i) to (vi) or (viii) variants of any one of claims (i) to (vii) 30 for increasing protein production activity in a eukaryotic host cell. 15/1 Said purified and isolated DNA sequence usually further comprises one or more regulatory sequences, as known in the art e.g. a promoter and/or an enhancer, polyadenylation sites and splice junctions usually employed for the expression of the protein or may optionally encode a selectable marker which is operably linked to a 5 gene of interest. The DNA sequences of this invention can be isolated according to standard PCR protocols and methods well known in the art. Promoters which can be used provided that such promoters are compatible with the host cell are, for example, promoters obtained from the genomes of viruses such as o polyoma virus, adenovirus (such as Adenovirus 2), papilloma virus (such as bovine papilloma virus), avian sarcoma virus, cytomegalovirus (such as murine or human cytomegalovirus immediate early promoter), a retrovirus, hepatitis-B virus, and Simian heterologous mammalian promoters, such as the actin promoter or an immunoglobulin promoter or heat shock promoters. Such regulatory sequences 5 direct constitutive expression. Furthermore, the purified and isolated DNA sequence might further comprise regulatory sequences which are capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in 0 the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBOJ. 8: 729-733) and immunoglobulins (Banjeri, et al., 1983. Cell 33: 729-740; Queen and 25 Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912 916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application No.264,166). 15/2 Developmentally-regulated promoters are also encompassed. Examples of such promoters include, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and thea-fetoprotein promoter (Campes and Tilghman, 1989. Genes 15/3 WO 2005/040377 PCT/EP2004/011974 Dev. 3: 537-546). Regulatable gene expression promoters are well known in the art, and include, by way of non-limiting example, any promoter that modulates expression of a gene encoding a 5 desired protein by binding an exogenous molecule, such as the CRE/LOX system, the TET system, the doxycycline system, the NFkappaB/UV light system, the Leu3p/isopropylmalate system, and theGLVPc/GAL4 system (See e. g., Sauer, 1998, Methods 14 (4): 381-92 ; Lewandoski, 2001, Nat. Rev. Genet 2 (10): 743-55; Legrand Poels et al., 1998, J. Photochem. Photobiol. B. 45: 18; Guo et al., 1996, FEBS Lett. 390 10 (2): 191-5; Wang et al., PNAS USA, 1999,96 (15): 84838). However, one skilled in the art would be able to determine other kinds of promoters that are suitable in carrying out the present invention. Enhancers can be optionally included in the purified DNA sequence of the invention 15 then belonging to the regulatory sequence, e.g. the promoter. The "gene of interest" or "transgene" preferably encodes a protein (structural or regulatory protein). As used herein "protein" refers generally to peptides and polypeptides having rnore than about ten amino acids. The proteins may be 20 "homologous" to the host (i.e., endogenous to the host cell being utilized), or "heterologous," (i.e., foreign to the host cell being utilized), such as a human protein produced by yeast. The protein may be produced as an insoluble aggregate or as a soluble protein in the periplasmic space or cytoplasm of the cell, or in the extracellular medium. Examples of proteins include hormones such as growth hormone or 25 erythropoietin (EPO), growth factors such as epidermal growth factor, analgesic substances like enkephalin, enzymes like chymotrypsin, receptors to hormones or growth factors, antibodies and include as well proteins usually used as a visualizing marker e.g. green fluorescent protein. 30 Preferably the purified DNA sequence further comprises at least a second isolated matrix attachment region (MAR) nucleotide sequence selected from the group comprising - a purified and isolated DNA sequence having protein production increasing activity, 35 - a purified and isolated MAR DNA sequence identifiable according to the method for identifying a MAR sequence using the described bioinformatic tool, the combined method or the method comprising at least one filter, - the sequences SEQ ID Nos 1 to 27, - a purified and isolated cLysMAR element and/or fragment, 40 - a synthetic MAR sequence comprising natural MAR element and/or fragments assembled betvveen linker sequences, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. The isolated matrix attachment reg ion (MAR) nucleotide sequence might be identical or different. 45 Alternatively, a first and a second identical MAR nucleotide sequence are used. Preferably, the MAR nucleotide sequences are located at both the 5' and the 3' ends of the sequence containing the promoter and the gene of interest. But the invention also envisions the fact that said first and or at least second MAR nucleotide sequences are 50 located on a sequence distinct from the one containing the promoter and the gene of interest. 16 WO 2005/040377 PCT/EP2004/011974 Embraced by the scope of the present invention is also the purified and isolated DNA sequence comprising a first isolated matrix attachment region (MAR) nucleotide sequence which is a MAR nucleotide sequence selected from the group comprising - a purified and isolated DNA sequence having protein production increasing 5 activity, - a purified and isolated MAR DNA sequence identifiable according to the method for identifying a MAR sequence using the described bioinformatic tool, the combined method or the method comprising at least one filter, - the sequences SEQ ID Nos 1 to 27, 10 - a purified and isolated cLysMAR element and/or fragment, - a synthetic MAR sequence comprising natural MAR element and/or fragments assembled between linker sequences, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants that can be 15 used for increasing protein production activity in a eukaryotic host cell by introducing the purified and isolated DNA sequence into a eukaryotic host cell according to well known protocols. Usually applied methods for introducing DNA into eukaryotic host cells applied are e.g. direct introduction of cloned DNA by microinjection or microparticle bombardment; electrotransfer ;use of viral vectors; encapsulation within a carrier 20 system; and use of transfecting reagents such as calcium phosphate, diethylaminoethyl (DEAE) -dextran or commercial transfection systems like the Lipofect-AMINE 2000 (Invitrogen). Preferably, the transfection method used to introduce the purified DNA sequence into a eukaryotic host cell is the method for transfecting a eukaryotic cell as described below. 25 The purified and isolated DNA sequence can be used in the form of a circular vector. Preferably, the purified and isolated DNA sequence is used in the form of a linear DNA sequence as vector. 30 As used herein, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used vector form. However, the invention is intended to include such other forms of expression vectors, including, but not limited to, viral vectors (e. g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. 35 The present invention further encompasses a method for transfecting a eukaryotic host cell, said method comprising a) introducing into said eukaryotic host cell at least one purified DNA sequence comprising at least one DNA sequence of interest and/or at least one purified 40 and isolated DNA sequence comprising a MAR nucleotide sequence or other chromatin modifying elements, b) subjecting within a defined time said transfected eukaryotic host cell to at least one additional transfection step with at least one purified DNA sequence comprising at least one DNA sequence of interest and/or with at least one 45 purified and isolated DNA sequence comprising a MAR nucleotide sequence or other chromatin modifying elements c) selecting said transfected eukaryotic host cell. Preferably at least two up to four transfecting steps are applied in step b). 50 In order to select the successful transfected cells, a gene that encodes a selectable marker (e. g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. The gene that encodes a selectable marker might be located 17 WO 2005/040377 PCT/EP2004/011974 on the purified DNA sequence comprising at least one DNA sequence of interest and/or at least one purified and isolated DNA sequence consisting of a MAR nucleotide sequence or other chromatin modifying elements or might optionally be co-introduced in separate form e.g. on a plasmid. Various selectable markers include those that confer 5 resistance to drugs, such as G418, hygromycin and methotrexate. The amount of the drug can be adapted as desired in order to increase productivity Usually, one or more selectable markers are used. Preferably, the selectable markers used in each distinct transfection steps are different. This allows selecting the 10 transformed cells that are "multi-transformed" by using for example two different antibiotic selections. Any eukaryotic host cell capable of protein production and lacking a cell wall can be used in the methods of the invention. Examples of useful mammalian host cell lines 15 include human cells such as human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol 36, 59 (1977)), human cervical carcinoma cells (HELA, ATCC CCL 2), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065); rodent cells such as baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub and 20 Chasin, Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)), mouse sertoli cells (TM4, Mather, Biot. Reprod 23, 243-251 (1980)), mouse mammary tumor (MMT 060562, ATCC CCL51); and cells from other mammals such as monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); canine kidney cells (MDCK, 25 ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); myeloma (e.g. NSO) /hybridoma cells. Preferably, the selected transfected eukaryotic host cells are high protein producer cells with a production rate of at least 10 pg per cell per day. Most preferred for uses herein are mammalian cells, more preferred are CHO cells. 30 The DNA sequence of interest of the purified and isolated DNA sequence is usually a gene of interest preferably encoding a protein operably linked to a promoter as described above. The purified and isolated DNA sequence comprising at least one DNA sequence of interest might comprise additionally to the DNA sequence of interest MAR 35 nucleotide sequence or other chromatin modifying elements. Purified and isolated DNA sequence comprising a MAR nucleotide sequence are for example selected from the group comprising the sequences SEQ ID Nos 1 to 27 and/or particular elements of the cLysMAR e.g. the B, K and F regions as well as fragment and 40 elements and combinations thereof as described above. Other chromatin modifying elements are for example boundary elements (BEs), locus control regions (LCRs), and universal chromatin opening elements (UCOEs) (see Zahn-Zabal et al. already cited). An example of multiple transfections of host cells is shown in Example 12 (Table 3). The first transfecting step (primary transfection) is carried out with the gene of interest 45 (SV40EGFP) alone, with a MAR nucleotide sequence (MAR) alone or with the gene of interest and a MAR nucleotide sequence (MAR-SV40EGFP). The second transfecting step (secondary transfection) is carried out with the gene of interest (SV40EGFP) alone, with a MAR nucleotide sequence (MAR) alone or with the gene of interest and a MAR nucleotide sequence (MAR-SV40EGFP), in all possible combinations resulting 50 from the first transfecting step. Preferably the eukaryotic host cell is transfected by: 18 WO 2005/040377 PCT/EP2004/011974 a) introducing a purified DNA sequence comprising one DNA sequence of interest and additionally a MAR nucleotide sequence, b) subjecting within a defined time said transfected eukaryotic host cell to at least one additional transfection step with the same purified DNA sequence comprising one DNA 5 sequence of interest and additionally a MAR nucleotide sequence of step a). Also preferably, the MAR nucleotide sequence of the of the purified and isolated DNA sequence is selected form the group comprising - a purified and isolated DNA sequence having protein production increasing 10 activity, - a purified and isolated MAR DNA sequence identifiable according to the method for identifying a MAR sequence using the described bioinformatic tool, the combined method or the method comprising at least one filter, - the sequences SEQ ID Nos 1 to 27, 15 - a purified and isolated cLysMAR element and/or fragment, - a synthetic MAR sequence comprising natural MAR element and/or fragments assembled between linker sequences, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. 20 Surprisingly, a synergy between the first and second transfection has been observed. A particular synergy has been observed when MAR elements are present at one or both of the transfection steps. Multiple transfections of the cells with pMAR alone or in combination with various expression plasmids, using the method described above have 25 been carried out. For example, Table 3 shows that transfecting the cells twice with the pMAR-SV40EGFP plasmid gave the highest expression of GFP and the highest degree of enhancement of all conditions (4.3 fold). In contrast, transfecting twice the vector without MAR gave little or no enhancement, 2.8-fold, instead of the expected two-fold increase. This proves that the presence of MAR elements at each transfection step is of 30 particular interest to achieve the maximal protein synthesis. As a particular example of the transfection method, said purified DNA sequence comprising at least one DNA sequence of interest can be introduced in form of multiple unlinked plasmids, comprising a gene of interest operably linked to a promoter, a selectable marker gene, and/or protein production increasing elements such as MAR 35 sequences. The ratio of the first and subsequent DNA sequences may be adapted as required for the use of specific cell types, and is routine experimentation to one ordinary skilled in the art. 40 The defined time for additional transformations of the primary transformed cells is tightly dependent on the cell cycle and on its duration. Usually the defined time corresponds to intervals related to the cell division cycle. Therefore this precise timing may be adapted as required for the use of specific cell 45 types, and is routine experimentation to one ordinary skilled in the art. Preferably the defined time is the moment the host cell just has entered into the same phase of a second or a further cell division cycle, preferably the second cycle. This time is usually situated between 6h and 48 h, preferably between 20h and 24h after the previous transfecting event. 50 Also encompassed by the present invention is a method for transfecting a eukaryotic host cell, said method comprising co-transfecting into said eukaryotic host cell at least one first purified and isolated DNA sequence comprising at least one DNA sequence of 19 WO 2005/040377 PCT/EP2004/011974 interest, and a second purified DNA comprising at least one MAR nucleotide selected from the group comprising: - a purified and isolated DNA sequence having protein production increasing activity, 5 - a purified and isolated MAR DNA sequence identifiable according to the method for identifying a MAR sequence using the described bioinformatic tool, the combined method or the method comprising at least one filter, - the sequences SEQ ID Nos I to 27, - a purified and isolated cLysMAR element and/or fragment, 10 - a synthetic MAR sequence comprising natural MAR element and/or fragments assembled between linker sequences, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. Said first purified and isolated DNA sequence can also comprise at least one MAR 15 nucleotide as described above. Also envisioned is a process for the production of a protein wherein a eukaryotic host cell is transfected according to the transfection methods as defined in the present invention and is cultured in a culture medium under conditions suitable for expression of the protein. Said protein is finally recovered according to any recovering process known 20 to the skilled in the art. Given as an example, the following process for protein production might be used. The eukaryotic host cell transfected with the transfection method of the present invention is used in a process for the production of a protein by culturing said cell under 25 conditions suitable for expression of said protein and recovering said protein. Suitable culture conditions are those conventionally used for in vitro cultivation of eukaryotic cells as described e.g. in WO 96/39488. The protein can be isolated from the cell culture by conventional separation techniques such as e.g. fractionation on immunoaffinity or ion-exchange columns; precipitation; reverse phase HPLC; 30 chromatography; chromatofocusing; SDS-PAGE; gel filtration. One skilled in the art will appreciate that purification methods suitable for the polypeptide of interest may require modification to account for changes in the character of the polypeptide upon expression in recombinant cell culture. 35 The proteins that are produced according to this invention can be tested for functionality by a variety of methods. For example, the presence of antigenic epitopes and ability of the proteins to bind ligands can be determined by Western blot assays, fluorescence cell sorting assays, immunoprecipitation, immunochemical assays and/or competitive binding assays, as well as any other assay which measures specific binding 40 activity. The proteins of this invention can be used in a number of practical applications including, but not limited to: 1. Immunization with recombinant host protein antigen as a viral/pathogen antagonist. 45 2. Production of membrane proteins for diagnostic or screening assays. 3. Production of membrane proteins for biochemical studies. 4. Production of membrane protein for structural studies. 5. Antigen production for generation of antibodies for immuno-histochemical mapping, including mapping of orphan receptors and ion channels. 50 Also provided by the present invention is a eukaryotic host cell transfected according to any of the preceding transfection methods. Preferably, the eukaryotic host cell is a mammalian host cell line. 20 WO 2005/040377 PCT/EP2004/011974 As already described, example of useful mammalian host cell lines include human cells such as human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol 36, 59 (1977)), human cervical carcinoma cells (HELA, ATCC CCL 2), human lung cells (W138, ATCC CCL 75), 5 human liver cells (Hep G2, HB 8065); rodent cells such as baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Nat/. Acad. Sci. USA, 77, 4216 (1980)), mouse sertoli cells (TM4, Mather, Biol: Reprod 23, 243-251 (1980)), mouse mammary tumor (MMT 060562, ATCC CCL51); and cells from other mammals such as monkey kidney CV1 line transformed by SV40 10 (COS-7, ATCC CRL 1651); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); myeloma (e.g. NSO) /hybridoma cells. Most preferred for uses herein are CHO cells. 15 The present invention also provides for a cell transfection mixture or Kit comprising at least one purified and isolated DNA sequence according to the invention. The invention further comprises a transgenic organism wherein at least some of its cells 20 have stably incorporated at least one DNA sequence of - a purified and isolated DNA sequence having protein production increasing activity, - a purified and isolated MAR DNA sequence identifiable according to the method for identifying a MAR sequence using the described bioinformatic tool, the 25 combined method or the method comprising at least one filter, - the sequences SEQ ID Nos 1 to 27, - a purified and isolated cLysMAR elernent and/or fragment, - a synthetic MAR sequence comprising natural MAR element and/or fragments assembled between linker sequences, 30 a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. Preferably, some of the cells of the transgenic organisms have been transfected according the methods described herein. 35 Also envisioned in the present invention is a transgenic organism wherein its genome has stably incorporated at least one DNA sequence of - a purified and isolated DNA sequence having protein production increasing activity, - a purified and isolated MAR DNA sequence identifiable according to the method 40 for identifying a MAR sequence using the described bioinformatic tool, the combined method or the method comprising at least one filter, - the sequences SEQ ID Nos I to 27, - a purified and isolated cLysMAR element and/or fragment, - a synthetic MAR sequence comprising natural MAR element and/or fragments 45 assembled between linker sequences, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof and variants. 50 Transgenic eukaryotic organisms which can be useful for the present invention are for example selected form the group comprising mammals (mouse, human, monkey etc) and in particular laboratory animals such as rodents in general, insects (drosophila, 21 etc), fishes (zebra fish, etc.), amphibians (frogs, newt, etc..) and other simpler organisms such as c. elegans, yeast, etc.. Yet another object of the present invention is to provide a computer readable medium comprising computer-executable instructions for performing the method for 5 identifying a MAR sequence as described in the present invention. The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention. o Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Each document, reference, patent application or patent cited in this text is expressly 5 incorporated herein in their entirely by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application, or patent cited in this text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be 20 understood as a concession that the material or information was part of the common general knowledge or was known in Australia or any other country. 22 WO 2005/040377 PCT/EP2004/011974 EXAMPLES Example 1: SMAR Scan@ and MAR sequences 5 A first rough evaluation of SMAR Scan@ was done by analyzing experimentally defined human MARs and non-MAR sequences. As MAR sequences, the previous results from the analysis of human MARs from SMARt Db were used to plot a density histogram for each criterion as shown in Fig. 1. Similarly, non-MAR sequences were also analyzed 10 and plotted. As non-MAR sequences, all Ref-Seq-contigs from the chromosome 22 were used, considering that this latter was big enough to contain a negligible part of MAR sequences regarding the part of non-MAR sequences. The density distributions shown in Fig. I are all skewed with a long tail. For the highest 15 bend, the highest major groove depth and the highest minor groove width, the distributions are right skewed. For the lowest melting temperature, the distributions are left-skewed which is natural given the inverse correspondence of this criterion regarding the three others. For the MAR sequences, biphasic distributions with a second weak peak, are actually apparent. And between MAR and non-MAR sequences distributions, 20 a clear shift is also visible in each plot. Among all human MAR sequences used, in average only about 70% of them have a value greater than the 75th quantile of human MARs distribution, this for the four different criteria. Similarly concerning the second weak peak of each human MARs 25 distribution, only 15% of the human MAR sequences are responsible of these outlying values. Among these 15% of human MAR sequences, most are very well documented MARs, used to insulate transgene from position effects, such as the interferon locus MAR, the beta-globin locus MAR (Ramezani A, Hawley TS, Hawley RG, "Performance and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold 30 attachment region and the chicken beta-globin insulator", Blood, 101:4717-4724, 2003), or the apolipoprotein MAR (Namciu, S, Blochinger KB, Fournier REK, "Human matrix attachment regions in-sulate transgene expression from chromosomal position effects in Drosophila melanogaster", Mol. Cell. Biol., 18:2382-2391, 1998). Always with the same data, human MAR sequences were also used to determine the 35 association between the four theoretical structural properties computed and the AT content. Fig. 2 represents the scatterplot and the corresponding correlation coefficient r for every pair of criteria. Example 2: Distribution plots of MAR sequences by organism 40 MAR sequences from SMARt DB of other organisms were also retrieved and analyzed similarly as explained previously. The MAR sequences density distributions for the mouse, the chicken, the sorghum bicolor and the human are plotted jointly in Fig. 3. 45 Example 3: MAR prediction of the whole chromosome 22 All RefSeq contigs from the chromosome 22 were analyzed by SMAR Scan@ using the default settings this time. The result is that SMAR Scan® predicted a total of 803 MARs, their average length being 446 bp, which means an average of one MAR 50 predicted per 42 777 bp. The total length of the predicted MARs corresponds to I % of the chromosome 22 length. The AT-content of the predicted regions ranged from 23 WO 2005/040377 PCT/EP2004/011974 65,1% to 93.3%; the average AT-content of all these regions being 73.5%. Thus, predicted MARs were AT-rich, whereas chromosome 22 is not AT-rich (52.1% AT). SMARTest was also used to analyze the whole chromosome 22 and obtained 1387 5 MAR candidates, their average length being 494 bp representing an average of one MAR predicted per 24 765 bp. The total length of the predicted MARs corresponds to 2% of the chromosome 22. Between all MARs predicted by the two softwares, 154 predicted MARs are found by both programs, which represents respectively 19% and 11 % of SMAR Scan@ and SMARTest predicted MARs. Given predicted MARs mean 10 length for SMAR Scan@ and SMARTest, the probability to have by chance an overlapping between SMAR Scan@ and SMARTest predictions is 0.0027% per prediction. To evaluate the specificity of SMAR Scan@ predictions, SMAR Scan@ analyses were 15 performed on randomly shuffled sequences of the chromosome 22 (Fig. 4). Shuffled sequences were generated using 4 different methods: by a segmentation of the chromosome 22 into ndnoverlapping windows of 10 bp and by separately shuffling the nucleotides in each window; by "scrambling" which means a permutation of all nucleotides of the chromosome; by "rubbling" which means a segmentation of the chro 20 mosome in fragments of 10 bp and a random assembling of these fragments and finally by order 1 Markov chains, the different states being the all the different DNA dinucleotides and the transition probabilities between these states being based on the chromosome 22 scan. For each shuffling method, five shuffled chromosome 22 were generated and analyzed by SMAR Scan@ using the default settings. Concerning the 25 number hits, an average of 3 519 170 hits (sd: 18 353) was found for the permutated chromosome 22 within nonoverlapping windows of 10 bp, 171 936,4 hits (sd: 2 859,04) for the scrambled sequences and 24 708,2 hits (sd: 1 191,59) for the rubbled chromosome 22 and 2 282 hits in average (sd: 334,7) for the chromosomes generated according to order I Markov chains models of the chromosome 22, which respectively 30 represents 185% (sd: 0.5% of the mean), 9% (sd: 1.5%), 1% (sd: 5%) and 0.1% (sd: 15%) of the number of hits found with the native chromosome 22. For the number of MARs predicted, which thus means contiguous hits of length greater than 300, 1 997 MARs were predicted with the shuffled chromosome 22 within windows of 10 bp (sd: 31.2), only 2.4 MARs candidates were found in scrambled sequences (sd: 0.96) and 35 none for the rubbled and for the sequences generated according to Markov chains model, which respectively represents 249% and less than 0.3% of the number of predicted MARs found with the native chromosome 22. These data provide indications that SMAR Scan@ detects specific DNA elements which organization is lost when the DNA sequences are shuffled . 40 Example 4: Analysis of known matrix attachment regions in the Interferon locus with SMAR Scan@ 45 The relevance of MAR prediction by SMAR Scan@ was investigated by analyzing the recently published MAR regions of the human interferon gene cluster on the short arm of chromosome 9 (9p22). Goetze et al. (already cited) reported an exhaustive analysis of the WP1 8A1 0A7 locus to analyze the suspected correlation between BURs (termed in this case stress-induced duplex destabilization or SIDD) and in vitro binding to the 50 nuclear matrix (Fig. 9, lower part). Three of the SIDD peaks were in agreement with the in vitro binding assay, while others did not match matrix attachment sites. Inspection of the interferon locus with SMAR Scan@ (Fig. 9, top part) indicated that three majors peaks accompanied by clusters of SATB1, NMP4 and MEF2 regulators binding sites 24 WO 2005/040377 PCT/EP2004/011974 correlated well with the active MARs. Therefore, we conclude that the occurrence of predicted CUEs and binding sites for these transcription factors is not restricted to the cLysMAR but may be a general property of all MARs. These results also imply that the SMAR Scan@ program efficiently detects MAR elements from genomic sequences. 5 Example 5: Accuracy of SMAR Scan@ prediction and comparison with other predictive tools The accuracy of SMAR Scan@ was evaluated using six genomic sequences for which 10 experimentally determined MARs have been mapped. In order to perform a comparison with other predictive tools, the sequences analyzed are the same with the sequences previously used to compare MAR-Finder and SMARTest. These genomic sequences are three plant and three human sequences (Table 1) totalizing 310 151 bp and 37 experimentally defined MARs. The results for SMARTest and MAR-Finder in Table 1 15 come from a previous comparison (Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I and Werner T, In silico pre-diction of scaffold/matrix attachment regions in large genomic sequences, Genome Research, 12:349-354, 2001.). MAR-Finder has been used with the default parameters excepted for the threshold that has been set to 0.4 and for the analysis of the protamine locus, the AT-richness 20 rule has been excluded (to detect the non AT-rich MARs as was done for the protamine locus). 25 WO 2005/040377 PCT/EP2004/011974 Sequence, description Length lxperiment- SMARTest MAR-inder SMIPAR San and reference ally defined prediction prediction prediction MARs positions positions positions positions (kb) (k(b) (kb) (kb) (kb) Cryza Sativa putative 30.034 0.0-1.2 ADP-glucose pyro- 5.4-7.4 6.-7.0 phosphorylase subunit 15.2-i5.? 15.7-15.9 15-6-16 SH2 and putative 16.2-16.6 NADPH dependant 17.3-18.5 17.648.3 17.5-12.4 M.6-182 reductase Al genes 20.0-23.1 19.6-20.1 19,820.4 21.. 6-22 (U705411. [4] 207-21.3 21.3-21.5 23.6-23.9 23.9-24.2 23.4-23.8 25.0-25.4 24.7-25. 1 27.5-27.9 SorghmLn bicolor ADP- 42.446 0.0-1.5 - glucose pyrphoopho- 7.1-97 - - 74-73 iyiase subun it SH2, 21.3-21,9 - 215-21.8 NADPH-lependant 22.4-24.7 22.9-24.0 23.2-24.2 22.9-23.2 reducatse Al-b genes - - 23.6-24,0 (AFO10283), [4] 27.3-27.6 26.9-27.5 273-27.6 32.5-33.7 - - 334-33.9 41.6-42.3 - Sorghum bicolor BAC 7.195 0.9 clone 10K5 ~5.8 - (AF1 2404,), [37] 6.3 - ~-15.0 15.1-15.8 v21.9 21.7-22.0 - 214-21.0 ~23.3 ~25.6 - - - 29.2-29.5 39.0-40.0 ~44.1 44.1-44.5 48.5 47.9-49.5 47.9-494 48.1-48.6 48.2-493 -57.9 - - 62.9 63.1-63.7 >67.1 - 69.3 ~,-73.7 74.3-74.7 743-74.6 Human alpha-1-antitry- 30.461 2.6-6.3 5.5-6.0 3.0-3.2 .4-5.8 sin and corticosteroid - 5.1-t.0 binding globulin 22.0-30.4 25-26.2 24.9-25.3 252-26.4 intergenic region 27.5.27.8 25.5-25. (AF1 56545), [35] 262-26. -27.5-2:.2 Human protamine locus 53.050 W&9.7 (U 15422). [24] 32.6-33.6 - 33,9-34.8 37.2-39.4 - 33.9-34.8 51.8-53.0 - Human beta-globin 75.955 1.5-1.0 - - 2.3-26 locus 15.6-19.0 18.0-184 15.5-16,0 15.3-15.6 (UO1317), [21] - 18.0-18.4 34.4-34.9 44.7-52.7 - 50.6-50;8 56.6-57.1 56.5-57.2 60.0-70.0 59.8-60.3 58.1-58.5 62.8-63.1 65.6-66.0 63.0-63.6 26 WO 2005/040377 PCT/EP2004/011974 67.667.9 69.7-69.3 66.3.867 Sum(kIb 310.151 at. least 5b.1 14.5 1.8 9.5 Total numbI 11)ers :37 28 25 22 Average kb !prelicted 11.076 12.406 14.097 MA R True p-ositives [nu1.mber 19[14} 20['12] '17[14] of experimentally defined MAR foun]l False positives 9 5 5 False negatives 23 25 23 Specificity 19/28= 68% /25= 80% 17/22= 77% Sensitivity 14/37= 38% 12/37= 32% 1 4!37= 38% Table 1: Evaluation of SMAR Scan@ accuracy 5 Six different genomic sequences, three plant and three human sequences, for which experimentally defined MARs are known, were analyzed with MAR-Finder, SMARTest and SMAR Scan@. True positive matches are printed in bold, minus (-) indicates false negative matches. Some of the longer experimentally defined MARs contained more than one in silico prediction, each of them was counted as true positive match. 10 Therefore, the number of true in silico predictions is higher than the number of experimentally defined MARs found. Specificity is defined as the ratio of true positive predictions, whereas sensitivity is defined as the ratio of experimentally defined MARs found. * AT-rich rule excluded using MAR-Finder. 15 SMARTest predicted 28 regions as MARs, 19 (true positives) of these correlate with experimentally defined MARs (specificity: 68%) whereas 9 (32%) are located in non MARs (false positives). As some of the longest experimentally determined MARs contains more than one in silico prediction, the 19 true positives correspond actually to 14 different experimentally defined MARs (sensitivity: 38%). MARFinder 20 predicted 25 regions as MARs, 20 (specificity: 80%) of these correlate with experimentally defined MARs corresponding to 12 different experimentally defined MARs (sensitivity: 32%). SMAR Scan@ predicted 22 regions, 17 being true positives (specificity: 77%) matching 14 different experimentally defined MARs (sensitivity: 38%). 25 As another example, the same analysis has been applied to human chromosomes 1 and 2 and lead to the determination of 23 MARs sequences (SEQ ID N* I to 23). These sequences are listed in Annex 1 in ST25 format. Example 6: Analyses of the whole genome using the combined method (SMAR 30 Scan@-pfsearch) In order to test the potential correlation between the structural features computed by SMAR Scan@ and the S/MAR functional activity, the whole human genome has been analyzed with the combined method with very stringent parameters, in order to get 35 sequences with the highest values for the theoretical structural features computed, which are called "super" S/MARs below. This was done with the hope to obtain predicted MAR elements with a very potential to increase transgene expression and recombinant protein production. The putative S/MARs hence harvested were first analyzed from the bioinformatics perpective in an attempt to characterize and classify 40 them. 27 WO 2005/040377 PCT/EP2004/011974 6.1 S/MARs predicted from the analysis of the whole human genome As whole human genome sequence, all human RefSeq (National Center for Biotechnology Information, The NCBI handbook [Internet]. Bethesda (MD): National 5 Library of Medicine (US), Oct. Chapter 17, The Reference Sequence (RefSeq) Project, 2002 (Available from http://www.ncbi.nih.gov/entrez/query.fcgi?db=Books) contigs (release 5) were used and analyzed with the combined method, using SMAR Scan® as filter in the first level processing, employing default settings except for the highest bend cutoff value, whereas a stringent threshold of 4.0 degrees (instead of 3.202 degrees) 10 has been used for the DNA bending criterion. In the second level processing, predicted transcription factors binding have been sought in the sequences selected from the previous step without doing any filtering on these sequences. 15 The analysis by the combined method of the whole human genome came up with a total of 1757 putative "super" S/MARs representing a total of 1 065 305 bp (0.35% of the whole human genome). Table 2 shows for each chromosome: its size, its number of genes, its number of S/MARs predicted, its S/MARs density per gene and its kb per 20 S/MAR. This table shows that there are very various gene densities per S/MAR predicted for the different chromosomes (standard deviation represents more than 50% of the mean of the density of genes per S/MAR predicted and the fold difference between the higher and the lower density of genes per S/MAR is 6,5). Table 2 also shows that the kb per S/MAR varies less that the density of genes per S/MAR (standard 25 deviation represents 25% of the mean of kb per S/MAR and the fold difference between the higher and the lower kb per S/MAR is 3.2). Chromosome Number of Size of the Number of Density of Kb per genes per chromosome S/MARs genes S/MAR chromosome (millions bp) predicted per S/MAR 1 2544 230 85 29.9 2705 2 1772 241 143 12.3 1685 3 1406 198 101 13.9 1960 4 1036 190 118 8.7 1610 5 1233 180 116 10.6 1551 6 1247 170 94 13.2 1808 7 1383 160 179 7.7 1754 8 942 145 77 12.2 1883 9 1100 119 48 22.9 2479 10 1003 133 71 14.1 1873 11 1692 132 67 25.2 1970 12 1278 131 78 16.3 1679 13 506 97 70 7.2 1385 14 1168 88 36 32.4 2444 15 895 83 35 25.5 2371 16 1107 81 41 27 1975 17 1421 80 37 38.4 2162 18 396 75 51 7.7 1470 19 1621 56 36 45.02 1555 20 724 60 28 25.8 2142 21 355 34 18 19.7 1888 22 707 34 28 25.2 1214 X 1168 154 170 6.8 905 Y 251 25 30 8.3 833 Sum 26955 3050 1 757 457 43312 Mean 1 123 127 73 19 1 804 Sd 510 72.8 45 10 462 30 Table 2: Number of S/MARs predicted per chromosome. The number of genes per chromosome 28 WO 2005/040377 PCT/EP2004/011974 corresponds to the NCBI human genome statistics (Build 34 Version 3) (National Center for Biotechnology Information, The NCBI handbook [Internet]. Bethesda (MD): National Library of Medicine (US), Oct. Chapter 17, The Reference Sequence (RefSeq) Project, 2002 (Available from http://www.ncbi.nih.gov/entrez/query.fcgi?db=Books) based on GenBank annotations. 5 Chromosome sizes are the sum of the corresponding human RefSeq (National Center. for Biotechnology Information, The NCBI handbook [Internet]. Bethesda (MD): National Library of Medicine (US), Oct. Chapter 17, The Reference Sequence (RefSeq) Project, 2002 (Available from http://www.ncbi.nih.gov/entrez/query.fcgi?db=Books) (release 5) contig lengths 10 6.2 Bioinformatics analysis of "super" MARS for transcription factor binding sites The 1757 predicted "super" S/MARs sequences obtained previously by SMAR Scan@ were then analyzed for potential transcription factors binding sites. This has been achieved using RMatch Professional (Kel AE, Gossling E, Reuter 1, Cheremushkin E, 15 KelMargoulis OV, Wingender E, MATCH: A tool for searching transcription factor binding sites in DNA sequences, Nucleic Acids Res. 31(13):35769, 2003), a weight matrixbased tool based on TRANSFAC (Wingender E, Chen X, Fricke E, Geffers R, Hehl R, Liebich I, Krull M, Matys V, Michael H, Ohnhauser R, Pruss M, Schacherer F, Thiele S, Urbach S, The TRANSFAC system on gene expression regulation, Nucleic 20 Acids Research , 29(1):2813, 2001), Match 2.0 Professional has been used with most of the default settings Match analysis was based on TRANSFAC Professional, release 8.2 (20040630). The sums of all transcrption factors binding prediction on the 1757 sequences analyzed according to Match are in Table 3. Based on this table, only the transcription factors totalizing at least 20 hits over the 1757 sequences 25 analyzed were considered for further analyses. Hereafter are some of the human transcription factors that are the most often predicted to bind on the 1757 putative S/MAR sequences and their Match description: Cdc5 (cell division control protein 5) a transcriptional 30 regulator/repressor, Nkx3A a homeodomain protein regulated by androgen, POU F1 (pituitaryspecific positive transcription factor 1) which is specific to the pituitary and stimulates cells proliferation. Thus, in addition to SATBI, NMP4 and MEF2, other transcription factors can participate in the activity of MARs. API 1 AR 2 Bach2 1 Brn2 1 C/EBP 20 C/EBPgamma 5 CDP CR3 1 COMP1 2 CREBP1 34 Cdc5 858 Cdx2 35 Evil 472 FOX 78 FOXD3 79 FOXJ2 244 FOXP3 29 Freac7 272 GATA1 2 GATA3 142 GATA4 125 HFH1 12 HFH3 1 HLF 275 HNF1 337 HNF3alpha 23 HNF3beta 71 HP1 2 Lhx3 22 MEF2 114 MRF2 57 Myc 18 NKX3A 849 Nkx25 2 Oct1 191 PBX 5 POU1F1 483 POU3F2 11 POU6F1 29 Pax3 3 Pax6 20 Piti 505 SRF 8 TEF 2852 TFIIA 14 TTF1 1 V$MTATAB 4 VBP 53 Vmw65 1 XFD1 65 XFD2 418 XFD3 2 35 29 WO 2005/040377 PCT/EP2004/011974 Table 3 is a summary of all transcription factors binding prediction (totalizing 20 hits or more) on the 1757 sequences analyzed. 5 6.3 Bioinformatics analysis of predicted "super" MARs for dinucleotide frequencies Various computer analysis were performed in order to easily identify "super" S/MAR sequences using an explicit criterion that could be identified without computing. Among those, a di-nucleotide analysis was performed on the 1757 superMARs, computing 10 each of the 16 possible dinucleotide percentage for each sequence considering both strands in the 5' > 3' direction. A summary (min., max., median, mean, 25th percentile and 75th percentile) as well as the histograms of each dinucleotide percentage over the 1757 S/MAR sequences are respectively presented in Table 4. A similar analysis was performed on randomly 15 selected sequences from the human genome, representing randomly selected non S/MAR sequences (which might however contain some MARs). Table 5 represents respectively a summary of the dinucleotide content analysis for these sequences. Table 4: Dinucleotide percentages over the 1757 S/MAR sequences 20 AA % AC % AG % AT % Minimum 0.000 0.0000 0.0000 18.50 25th percentile 4.234 0.9372 0.1408 32.11 Median 7.843 2.2408 0.4777 34.68 Mean 7.184 3.2117 1.0865 34.32 75th percentile 10.110 4.7718 1.5096 36.94 Maximum 17.290 12.9479 8.1230 50.00 CA% CC% CG% CT% Minimum 0.0000 0.00000 0.0000 0.0000 25th percentile 0,9695 0.00000 0.0000 0.1408 Median 1.9776 0.00000 0.0000 0.4777 Mean 2.6977 0.14123 0.2709 1.0865 75th percentile 3.7543 0.09422 0.1256 1.5096 Maximum 10.4061 4.24837 7.4410 8.1230 GA% GC% GG% GT% Minimum 0,00000 0.0000 0.00000 0.0000 25th percentile 0.08696 0.0000 0.00000 0.9372 Median 0.32616 0.0000 0.00000 2.2408 Mean 0.63347 0.2104 0.14123 3.2117 75th percentile 0.83333 0.1914 0.09422 4.7718 Maximum 5.77889 9.8795 4.24837 12.9479 TA% TC% TG% TT% Minimum 28.63 0.00000 0.0000 0.000 25th percentile 33.48 0.08696 0.9695 4.234 Median 35.22 0.32616 1.9776 7.843 Mean 35.29 0.63347 2.6977 7.184 75th percentile 37.14 0.83333 3.7543 10.110 Maximum 50.00 5.77889 10.4061 17.290 Considering the results of the predicted S/MAR elements and of the nonS/MAR se quences in the summary tables, noticeable differences can be noticed in the AT et TA 25 dinucleotide contents between these two groups of sequences. AT and TA represent respectively at least 18,5 % and 28.6 % of the dinucleotide content of the predicted S/MAR sequences, whereas the minimum percentages for the same dinucleotides in 30 WO 2005/040377 PCT/EP2004/011974 nonS/MAR sequences are respectively 0.3 % and 0%. Similarly, the maximum CC and GG content in S/MAR sequences is 4.2 %, whereas in nonS/MAR sequences the percentages for these two dinucleotides can amount up to 20.8 %. The correlation between AT and TA dinucleotide percentages and the DNA highest 5 bend as computed by SMAR Scan@ is depicted in Fig. 17 for the predicted S/MAR sequences and in Fig.18 for the nonS/MAR sequences. The different scatterplots of these figures show that the TA percentage correlates well with the predicted DNA bend as predicted by SMAR Scan@. 10 Table 5: Dinucleotide percentages over the 1757 nonS/MAR sequences summary AA % AC % AG % AT % Minimum 0.000 1.735 1.512 0.3257 25th percentile 7.096 4.586 6.466 5.1033 Median 9.106 5.016 7.279 6.8695 Mean 8.976 5.054 7.184 7.0108 75th percentile 10.939 5.494 7.969 8.7913 Maximum 17.922 13.816 12.232 23.1788 CA% CC% CG% CT% Minimum 3.571 0.8278 0.0000 1.512 25th percentile 6.765 4.1077 0.4727 6.466 Median 7.410 5.5556 0.8439 7.279 Mean 7.411 5.9088 1.2707 7.184 75th percentile 8.010 7.2460 1.5760 7.969 Maximum 15.714 20.8415 12.6074 12.232 GA% GC% GG% GT% Minimum 1.319 0.4967 0.8278 1.735 25th percentile 5.495 3.2615 4.1077 4.586 Median 6.032 4.4092 5.5556 5.016 Mean 6.065 4.7468 5.9088 5.054 75th percentile 6.602 5,8824 7.2460 5.494 Maximum 10.423 16.0000 20.8415 13.816 TA% TC% TG% TT% Minimum 0.000 1.319 3.571 0.000 25th percentile 3.876 5.495 6.765 7.096 Median 5.625 6.032 7.410 9.106 Mean 5.774 6.065 7.411 8.976 75th percentile 7.464 6.602 8.010 10.939 Maximum 24.338 10.423 15.714 17.922 Four of the novel super MARs were randomly picked and analyzed for AT and TA 15 dinucleotide content, and compared with the previously known chicken lysMAR, considering windows of 100 base pairs (Table 6). Surprinsigly, Applicants have shown that all of the super MARs have AT dinucleotide frequencies greater then 12%, and TA dinucleotides greater than 10% of the total dinucleotides analysed in a window of 100base pairs of DNA. The most efficient MARs 20 display values around 34% of the two dinucleotide pairs. Table 6. Summary of %AT and TA dinucleotide frequencies of experimentally verified MARs 25 CLysMAR (average of CUEs) AT%: 12.03 TA%: 10.29 SEQ ID No P1 68 AT% : 33.78 TA%: 33.93 SEQ ID No P1 6 AT%: 34.67 TA%: 34.38 SEQ ID No 31 WO 2005/040377 PCT/EP2004/011974 P1 42 AT%: 35.65 TA%: 35.52 SEQ ID No Mean value for all human "super"MARs AT%: 34.32 TA% : 35.29 Mean value for all human non-MARs AT% : 7.01 TA% : 5.77 6.4 Analysis of orthologous intergenic regions of human and mouse genomes 5 In order to get an insight on S/MAR evolution, orthologous intergenic regions of human and mouse genomes have been analysed with SMAR Scan@. The data set used is composed of 87 pairs of complete orthologous intergenic regions from the human and mouse genomes (Shabalina SA, Ogurtsov AY, Kondrashov VA, Kondrashov AS, Selective constraint in intergenic regions of human and mouse genomes, Trends 10 Genet, 17(7):3736, 2001) (average length -12 000 bp) located on 12 human and on 12 mouse chromosomes, the synteny of these sequences was confirmed by pairwise sequence alignment and consideration of the annotations of the flanking genes (experimental or predicted). 15 Analysis of the 87 human and mouse orthologous intergenic sequences have been analysed with SMAR Scan@ using its default settings. Analysis of the human sequences yielded a total of 12 S/MARs predicted (representing a total length of 4 750 bp), located on 5 different intergenic sequences. 20 Among the three human intergenic sequences predicted to contain a "super" S/MAR using SMAR Scan@ stringent settings, one of the corresponding mouse orthologous intergenic sequence is also predicted to contain a S/MAR (human EMBL ID: Z96050, position 28 010 to 76 951 othologous to mouse EMBL ID: AC015932, positions 59 884 to 89 963). When a local alignement of these two orthologous intergenic sequences is 25 performed, the best local alignement of these two big regions correspond to the regions predicted by SMAR Scan@ to be S/MAR element. A manual search for the mouse orthologs of the two other human intergenic sequences predicted to contain a "super" S/MAR was performed using the Ensembl Genome Browser (http://ensembl.org). The mouse orthologous intergenic sequences of these two human sequences were 30 retrieved using Ensembl orthologue predictions (based on gene names), searching the orthologous mouse genes for the pairs of human genes flanking these intergenic regions. Because SMAR Scan@ has been tuned for human sequences and consequently yields 35 little "super"MARs with mouse genomic sequences, its default cutoff values were slightly relaxed for the minimum size of contiguous hits to be considered as S/MAR (using 200 bp instead of 300 bp). Analysis by SMAR Scan@ of these mouse sequences predicted several S/MARs having high values for the different computed structural features. This finding suggests that the human MAR elements are conserved across 40 species. Example 7 : Dissection of the chicken lysozyme gene 5'- MAR The 3000 base pair 5'-MAR was dissected into smaller fragments that were monitored 45 for effect on transgene expression in Chinese hamster ovary (CHO) cells. To do so, seven fragments of -400 bp were generated by polymerase chain reaction (PCR). These PCR-amplified fragments were contiguous and cover the entire MAR sequence when placed end-to-end. Four copies of each of these fragments were ligated in a head-to-tail orientation, to obtain a length corresponding to approximately half of that of 32 WO 2005/040377 PCT/EP2004/011974 the natural MAR. The tetramers were inserted upstream of the SV40 promoter in pGEGFPControl, a modified version of the pGL3Control vector (Promega). The plasmid pGEGFPControl was created by exchanging the luciferase gene of pGL3Control for the EGFP gene from pEGFP-N1 (Clontech). The 5'-MAR-fragment-containing plasmids 5 thus created were co-transfected with the resistance plasmid pSVneo in CHO-DG44 cells using LipofectAmine 2000 (Invitrogen) as transfection reagent, as performed previously (Zahn-Zabal, M., et al., "Development of stable cell lines for production or regulated expression using matrix attachment regions" J Biotechnol, 2001. 87(1): p. 29-42.). After selection of the antibiotic (G-418) resistant cells, polyclonal cell 10 populations were analyzed by FACS for EGFP fluorescence. Transgene expression was expressed at the percentile of high expressor cells, defined as the cells which fluorescence levels are at least 4 orders of magnitude higher than the average fluorescence of cells transfected with the pGEGFPControl vector without MAR. 15 Fig. 5 shows that multimerized fragments B, K and F enhance transgene expression, despite their shorter size as compared to the original MAR sequence. In contrast, other fragments are poorly active or fully inactive. Example 8: Specificity of B, K and F regions in the MAR context 20 The 5'-MAR was serially deleted from the 5'-end (Fig.6, upper part) or the 3'-end (Fig.6, lower part), respectively. The effect of the truncated elements was monitored in an assay similar to that described in the previous section. Figure 6 shows that the loss of ability to stimulate transgene expression in CHO cells was not evenly distributed. 25 In this deletion study, the loss of MAR activity coincided with discrete region